Complementary microarray experiments to identify genes repressed by Zap1. A Venn diagram is shown describing our methods of identifying genes repressed by Zap1. Genes showing decreased expression in zinc-replete cells expressing a constitutive Zap1 allele (Zap1^TC) and in zinc-limited wild type (WT) cells were identified. E3, experiment 3; E4, experiment 4.

Confirmation of the microarray results for potential Zap1-repressed genes. A, S1 nuclease protection assays were performed using RNA isolated from cells grown under the same conditions as the two sets of microarray experiments. Controls for induced genes (ZRT1), repressed genes (ADH1 and ADH3), and for equal loading (calmodulin, CMD1) were included. In addition, candidate genes selected from Table 1 were also tested. The band intensities were quantified, and the fold changes are reported. These data confirmed the microarray results for these genes. E3, experiment 3; E4, experiment 4. B, zinc dose-dependent and Zap1-dependent repression of MET3, MET14 and MET16. Wild type (DY1457) cells were grown in LZM supplemented with a range of added zinc, and mRNA levels of MET3, MET14, MET16, and CMD1 were analyzed by S1 nuclease protection assay (lanes 1–4). In addition, wild type (WT) and zap1Δ mutant cells (ZHY6) were grown in low zinc (−Zn, LZM + 1 μm ZnCl₂), and RNA was isolated and analyzed by S1 nuclease protection assay (lanes 5 and 6).

MET30 has zinc- and Zap1-responsive gene expression. A, proposed regulatory circuitry controlling MET3, MET14, and MET16 expression in response to zinc. B, transcriptional regulation of MET30 in response to zinc and Zap1 activity. Wild type (DY1457) cells were grown in LZM supplemented with a range of added zinc, and mRNA levels of MET30 and CMD1 were analyzed by S1 nuclease protection assay (lanes 1–7). In addition, wild type and zap1Δ mutant cells (ZHY6) were grown in low zinc (−Zn, LZM + 1 μm ZnCl₂) (lanes 8 and 9), and wild type cells with and without expressing the constitutive Zap1^TC allele were grown in zinc-replete SD medium (+Zn, lanes 10 and 11). RNA was isolated and analyzed by S1 nuclease protection assay. C, met4Δ mutant strain (TAL31) was grown in LZM supplemented with methionine and with a range of added zinc. RNA was isolated and analyzed by S1 nuclease protection assay.

MET30 is a Zap1 direct target. A, diagram of the MET30 promoter. Met4 is recruited to this promoter by binding to the Cbf1, Met31, and Met32 proteins bound at the indicated sites. A potential ZRE is located upstream of these sites of Met4 recruitment. B, Zap1 binds specifically to the MET30 ZRE in vitro as assessed by electrophoretic mobility shift assay. Radiolabeled double-stranded oligonucleotides (0.5 pmol, 10,000 cpm) containing ZRE-like sequences from the indicated promoters were used as probes. The probes were mixed with 0 (−), 0.2 (±) or 0.4 μg (+) per reaction of purified Zap1 DNA binding domain (Zap1_DBD). FP denotes the free probe, and the arrow indicates the Zap1_DBD-DNA complex. The wild type TSA1 ZRE and mutant TSA1 ZRE (TSA1m) were included as positive and negative controls, respectively. The MET30 ZRE was used as probe in lanes 4 and 5. C, electrophoretic mobility shift assays were performed using the wild type TSA1 ZRE as the probe. Nonradiolabeled oligonucleotides were used as competitors at a 200-fold excess of the labeled probe concentration. Wild type TSA1 ZRE and MET30 ZRE fragments competed effectively for Zap1 binding, whereas the mutant TSA1m ZRE did not. D, MET30 ZRE is required for induction of gene expression in low zinc in vivo. Wild type (WT) or zap1Δ mutant cells bearing the pMET30-lacZ promoter fusion or the MET30 promoter fusion with a mutated ZRE (pMET30^mZRE-lacZ) were grown in high zinc (+Zn, LZM + 1000 μm ZnCl₂) and low zinc (−Zn, LZM + 1 μm ZnCl₂) for 14–20 h. Cells were then harvested and assayed for β-galactosidase activity. The vector YEp353 was used as negative control, and the Zap1-responsive pZRC1-lacZ served as a positive control. Shown are the means from three independent experiments, and the error bars indicate ± S.D.

Met4 protein level is regulated post-transcriptionally by zinc and by Zap1. A, immunoblot analysis of Met4 protein levels in wild type cells grown in LZM supplemented with a range of added zinc (lanes 2–6). A met4Δ strain (TAL31) was used as a control (lane 1). In addition, wild type and zap1Δ mutant cells (ZHY6) were grown in low zinc (−Zn, LZM + 1 μm ZnCl₂) (lanes 7 and 8) and wild type cells with and without expressing the constitutive Zap1^TC allele were grown in zinc-replete SD medium (+Zn, lanes 9 and 10). The multiple Met4 bands represent differences in ubiquitination and/or phosphorylation states of the protein. 3-Phosphoglycerate kinase (Pgk1) protein was used as a loading control. B, cells were grown under the same conditions as in A. RNA was isolated and was then analyzed by S1 nuclease protection assay. No change in MET4 mRNA was detected indicating the changes in protein level are because of post-transcriptional effects.

Decreased Met4 protein accumulation in zinc-limited cells requires the ubiquitination-proteasome degradation pathway. A, immunoblot analysis of Met4 protein from wild type (WT), pre1-1pre2-1, or hrd2-1 mutant cells. Cells were grown in high zinc (+, LZM + 1000 μm ZnCl₂) and low zinc (−, LZM + 1 μm ZnCl₂) prior to immunoblot analysis. Pgk1 is shown as a loading control. B, immunoblot analysis of Met4 protein levels from wild type (WT, DY1457) and pdr5Δ (CWY21) mutant cells grown in high zinc (+, LZM + 1000 μm ZnCl₂) and low zinc (−, LZM + 1 μm ZnCl₂) supplemented with MG132 (0.2 mg/ml in DMSO) or DMSO alone as a control. C, wild type Met4 and the Met4^K163R allele were expressed in a met4Δ strain (TAL31) from the GAL1 promoter using the GEV system (see “Experimental Procedures”). Cells were grown in high zinc (+, LZM + 1000 μm ZnCl₂) or low zinc (−, LZM + 1 μm ZnCl₂) in the presence of the inducer, β-estradiol, prior to immunoblotting. D, immunoblot analysis of Met4 protein levels in wild type cells grown in high zinc (+, LZM + 1000 μm ZnCl₂) or low zinc (−, LZM + 1 μm ZnCl₂) and in cells with or without the Zap1^TC allele in high zinc (LZM + 1000 μm ZnCl₂). The cells were treated with 1 mm cadmium for the indicated times prior to harvest. The cells in lanes 3, 9, and 10 were treated with cadmium for 8 h prior to harvest.

Derepression of MET3, MET14, and MET16 expression in cells with disrupted ubiquitination and proteasome degradation of Met4. A, wild type (WT, DY1457) and pdr5Δ (CWY21) mutant cells were grown in the same conditions as Fig. 6B. RNA was isolated and analyzed by S1 nuclease protection assay. CMD1 serves as a loading control. B, RNA samples for S1 nuclease protection assay were prepared from wild type (WT, DY1457) cells bearing the vector control and met4Δ cells (TAL31) expressing wild type Met4 or Met4^K163R as described in Fig. 6C.

Derepression of sulfate assimilation increases oxidative stress and the NADP⁺/NADPH ratio in zinc-limited cells. A, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were inoculated at low density (A₆₀₀ = 0.1) in high or low zinc media as described in Fig. 6C in the presence of the indicated concentration of H₂O₂. Cell densities were then measured after 38 h of culturing at 30 °C. Cell densities are reported as percent of the corresponding untreated controls. B, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in high or low zinc media as described in Fig. 6C and then assayed for reactive oxygen species using the fluorescent probe DCFH-DA. The values are means of three independent experiments, and the error bars represent ± S.D. C, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in low zinc medium (LZM + 1 μm ZnCl₂) prior to assay of total glutathione (Total GSH), reduced glutathione (GSH), and oxidized glutathione (GSSG). The ratios of GSSG/GSH ratios are shown in the right panel. The plotted values are the means of 12 independent cultures, and the error bars represent ±1 S.E. D, met4Δ mutant cells expressing wild type Met4 (WT) or Met4^K163R were grown in high or low zinc media as described in Fig. 6C prior to analysis of total NADP(H), reduced NADPH, and oxidized NADP⁺. The NADP⁺/NADPH ratio is reported in the right panel. The data plotted are the means of six independent cultures, and the error bars indicate 1 S.D. p values were determined using the paired, two-tailed Student's t test.

Role of Zap1 regulation in sulfate metabolism. S. cerevisiae genes whose expression is activated in low zinc are indicated in yellow, and genes negatively regulated in low zinc are indicated in blue. S. pombe genes up-regulated under zinc deficiency are marked in red, and those down-regulated in low zinc are denoted in green.